Introduction

Hematopoiesis is the process of creating a wide variety of blood and bone marrow cells, namely erythrocytes, platelets, granulocytes, lymphocytes, and monocytes. This process begins with multipotent hematopoietic stem cells (HSC) which have the capability of dividing into either a multipotent progenitor cell or to self-renew. Progenitor cells are then able to divide into increasing specialized cells, a process which repeats and eventually leads to mature white blood cells, red blood cells, or platelets.

This series of divisions in hematopoiesis creates a chart with several branch points beginning with the multipotent progenitor cells dividing into either a common myeloid progenitor or a common lymphoid progenitor. Common myeloid progenitors eventually go on to create megakaryocytes, erythrocytes, basophils, neutrophils, eosinophils, and monocytes. Common lymphoid progenitors will produce Natural Killer cells as well as B and T lymphocytes.[1]

Multi-potent progenitors and any cells the progenitors create lack the capacity to self-renew and must instead always divide into a further specialized cell. Which type of cell the HSCs and progenitor cells divide into is largely decided by the specific signaling factors such as erythropoietin, Interleukin (IL) -2, IL-3, IL-6, IL-7, a variety of colony stimulating factors, and the, niche that the HSC develops in.[2]

Issues of Concern

Novel cell lineage tracking technologies have led to recent revisions in the model of hematopoiesis. Classically, the model is as described above: HSCs divide into multipotent progenitor cells that can further divide into common myeloid progenitors or common lymphoid progenitors. Revised models demonstrate fewer branch points and instead show a steady differentiation of heterogenous HSC to the final cell lines. This leads to a to a more varied pool of HSC that are partially differentiated as opposed to groups of identical precursors. Traditionally these HSC are differentiated by cell surface markers which vary between HSC, progenitor cells, and mature cells. Cell surface markers between these partially differentiated HSC do not necessarily change despite a change in gene signaling, which gave rise to the classical branching model.[1]

Structure

The primary locations of hematopoiesis change throughout life. At the beginning of the fetal period, it begins in the yolk sac and aorta-gonad-mesonephros, eventually transitioning into liver, spleen, and finally the bone marrow and lymph nodes. It is maintained in these final locations for the duration of adult life except in pathological cases where it can return to its former sites.

Hematopoiesis in bone marrow takes place in islands of hematopoietic tissue surrounded by vascular sinuses and is interspersed with trabecular bone. Hematopoietic tissue contains a spectrum of blood cells, adipocytes, endothelial cells, and adventitial cells. As cells develop and mature, they enter the circulation through venous sinuses. The specific cells which are predominant in each microscopic area of the bone marrow are largely dependent on the niche of signaling cells at that site, which can include mature HSC, osteoblasts, macrophages, stromal cells, endothelial cells, and adipocytes among others. The location of hematopoiesis for certain cells can be tied to their function. For example, megakaryocytes, which are very large and bulky, are formed right next to the marrow sinuses into which they shed platelets.[3]

Regulation

Our understanding of the transcription factors and growth factors that govern hematopoiesis has continued to demonstrate the complexity of the system. Particular interest has been paid in determining the genes and factors that influence a malignant transformation. Selected examples of known important transcription factors include:

• T-cell acute leukemia-1 (TAL-1) which is necessary to keep multipotent stem cells multipotent and quiescent and is an essential regulator of hematopoiesis.  TAL-1 complexes with a variety of other transcription factors (E47/E2A, LMO2, GATA1–3, Ldb1/2, Ldb1, ETO, Runx1, ERG, FLI1) to determine differentiation into myeloid or lymphoid cells.[1][4]
• Bcl11a has been demonstrated to play an important role in the regulation of hematopoiesis both in the decision to proliferate and the specific cells to be made. Lack of Bcl11a expression was found to produce a signature of myeloid cells while reducing the signature genes associated with lymphoid development. It was also found that lack of expression was associated with cells in the S and G2/M phase, suggesting increased growth.[5]
• c-MYB: Primarily expressed in immature HSC, this helps to regulate fetal hemoglobin and consequently is a disease modifier of hemoglobinopathies and sickle cell. Dysregulation can lead to a variety of leukemias and lymphomas as well as solid tumors.[6]
• GATA-1: A zinc finger transcription factor that promotes the proliferation and differentiation of erythropoiesis both in the embryonic stage and the later definitive stages of production. Congenital disruption of this factor produces ineffective erythropoiesis.GATA-1, GATA-2, and GATA-3 are important in hematopoiesis.[1]

Other transcription factors such as Erg, Fli1, Tal1, Lyl1, Lmo2, Runx1 and Gata2 have been shown to work together as a complex to give rise to pre-HSC. Runx1, TEL/ETV6, SCL/TAL, and LMO2 mutations account for the majority of known leukemia-associated translocations.[7]

Histochemistry and Cytochemistry

Bone marrow aspiration or biopsies, as well as complete blood counts with differential, are used to look for issues with hematopoiesis. Typically these biopsies are taken from the iliac crest but may also be taken from the sternum, vertebrae, or tibia under certain conditions.

Stains of the biopsies are used to detect a variety of conditions including aplastic anemia, neoplasia, fibrosis, and inflammation. H and E staining allows practitioners to identify mature nearly mature cells of the various cell lines in hematopoietic tissue. Importantly, very immature cells including hematopoietic stem cells cannot be easily identified by this method as they are not easily discernable from one another and look more like lymphoblasts under the microscope. Typical immature myeloid cells are nucleated and more basophilic than their mature counterparts. Immature granulocytes will have large vesicular nuclei and will be less basophilic. Fluorescent staining for cell surface markers such as CD34 remains the key method for attempting to differentiate certain progenitor cells and undifferentiated HSC.[8][9]Flow Cytometry is also commonly used as a technique to differentiate cells including HSC at distinct points in hematopoiesis by their mRNA profiles. It can measure the different levels of various markers, monitoring cells through differentiation [10]

Microscopy, Light

Light microscopy of bone marrow aspirates is generally done using H and E staining.

Erythropoiesis under light microscopy demonstrates the spectrum of red cell differentiation from proerythroblasts to reticulocytes. The full process of differentiation can take up to 5 days. Proerythroblasts will become erythroblasts/basophilic normoblasts, which then become late normoblasts. As the cell matures, it becomes progressively smaller and with an increasingly condensed nucleus. After the late normoblast stage, the nucleus is expelled, and the cell is called a reticulocyte.  In times of anemia, this process may quicken leading to various debris visual on microscopy residing in the RBC. Examples include Heinz bodies, Pappenheimer bodies, and Howell-Jolly bodies.

Granulocyte production progresses from myeloblast to promyelocyte where it then branches into 1 of 3 precursors: basophilic myelocyte, neutrophilic myelocyte, and eosinophilic myelocyte. The precursors go on to form mature basophils, neutrophils, and eosinophils respectively. Owing to the overall predominance of neutrophils compared to these other cell lines neutrophilic precursors are seen much more commonly on light microscopy.

Megakaryocyte precursors are difficult to distinguish microscopically from myeloblasts. Further along in differentiation, they gain their distinctive cytoplasmic granulation and numerous nuclear lobes.

Early stages of lymphopoiesis do occur in the bone marrow but then shift largely to the peripheral lymphoid tissues. Plasma cells can be present in bone marrow but can be difficult to differentiate from other blast cells with light microscopy.

Microscopy, Electron

Electron microscopy shares many of the same limitations in identifying the specific hematopoietic cells with light microscopy. This technique is particularly useful in finding the locations of hematopoiesis in relation to the vascular sinuses and reticular fibers in the bone marrow.[3]

Clinical Significance

Extramedullary hematopoiesis is the term for any time hematopoiesis occurs outside of the bone marrow. This is normal in fetal development but may occur abnormally later in life in conditions which damage the bone marrow or promote hematopoiesis elsewhere. Examples include myelofibrosis of the bone marrow or high erythropoietin states such as hypoxia. In severe cases like those seen in untreated thalassemia, extramedullary hematopoiesis can lead to dangerous hepatomegaly and splenomegaly.[11]

Issues with hematopoiesis can lead to a variety of clinical conditions most prominently leukemias. Other selected examples include:

Polycythemia vera: A JAK2 mutation leads to erythroid precursors that over-respond to erythropoietin leading to increased RBC production. Clinically this can manifest as thromboses, pruritis, and skin discoloration.

Primary myelofibrosis:  A point mutation in the JAK2 tyrosine kinase is thought to lead to HSC and progenitor cells to be more sensitive to growth factors. This eventually causes scarring and fibrosis of the bone marrow and extramedullary hematopoiesis.

Essential thrombocythemia: JAK2 mutation leads to platelet overproduction. The clinical picture can involve bleeding, thromboses, migraines, and erythromelalgia.[11]

Hematopoietic stem cell transplant remains a key tool in the combat for a variety of conditions that are caused by or lead to improper hematopoiesis. Examples include leukemias, inherited blood disorders such as sickle cell disease or alpha/ beta thalassemia major, and potentially as a method of graft-versus-tumor treatment for other cancers.